A stroke is the rapidly developing loss of brain function(s) due to disturbance in the blood supply to the brain. This can occur due to a hemorrhage or due to ischemia (lack of oxygen and/or glucose supply) which may be caused, for example, by thrombosis or embolism. During ischemic stroke, blood flow to an area of the brain is blocked and, consequently, that area of the brain begins to cease function and will ultimately die. The initial area of neuronal death is called the ischemic core. Over time, irreversible injury will occur, often leading to death of the tissue, i.e. “infarction”. In the area of acute stroke research, considerable focus has been directed toward unraveling the mechanisms contributing to cellular necrosis resulting from prolonged ischemia in regions of the brain.
The return of blood flow to the ischemic area, known as “reperfusion”, may occur either spontaneously or as a result of a clot busting drug. Reperfusion is essential for the recovery of the brain area surrounding the ischemic core. However, in a detrimental and paradoxical response, the return of blood flow can increase cerebral edema and cause further brain damage, for example, through the introduction of free radicals (Dietrich, W. D. (1994) Morphological manifestations of reperfusion injury in brain. Ann NY Acad Sci, 723: 15-24); Aronowski, J. et al. (1997) Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats. J Cereb Blood Flow Met, 10: 1048-1056). This further damage is known as “reperfusion injury”.
Tissue-plasminogen-activator (tPA), a clot-busting drug, is the only clinically effective neuroprotectant currently administered following a stroke. However, tPA therapy is limited to administration within four hours of the onset of clinical signs of stroke, due to the detrimental effects of reperfusion injury (Kuroda, S. and B. K. Siesjo (1997) Reperfusion damage following focal ischemia pathophysiology and therapeutic windows. Clin Neurosci, 4: 199-212). It has been found that, after 4 hours, the damage due to reperfusion injury can exceed the damage caused by the prolonged ischemia.
Many pathological responses are involved in cerebral reperfusion injury, including but not limited to the introduction of superoxides, free radicals and leukocytes, platelet activation, and breakdown of the blood-brain barrier (Kamat C. et al. (2008). Antioxidants in central nervous system diseases: Preclinical promise and translational challenges. J Alzheimers Dis, 15: 473-493; Martin, R. L. (1997) Experimental neuronal protection in cerebral ischaemia Part 1: Experimental models and pathophysiological responses. J Clin Neuroscience, 4: 96-113).
Strategies to reduce or minimize cerebral reperfusion injury require an understanding of the various pathophysiological processes involved.
Various animal models have been developed in an attempt to study the effects of ischemia and/or reperfusion in various tissues, including animal models of ischemic stroke. The available models of ischemic stroke have limitations. One significant limitation of prior art models is high mortality rate during the experimental procedure. Another significant limitation of prior art models is a lack of reproducibility, and thus high variability, with respect to infarct size and/or location. A reproducible and focal ischemia is ideal. Thus, there is a long-felt need in the art for improved animal models having lower mortality rates and higher reproducibility (lower variability) compared to prior models.
Several prior animal models of ischemic stroke have examined the effects of permanent occlusion of the middle cerebral artery (MCA), one of three major paired arteries that supply blood to the cerebrum. For example, Robinson and colleagues studied permanent ligation of the MCA in rats (Robinson, R. G., et al. (1975) Nature, 255(5506): 332-334). Bederson and colleagues produced extensive (3 or 6 mm) occlusions of the MCA in rats by exposing the MCA transcranially and irreversibly occluding it with microbipolar coagulation (Bederson, J. B. et al. (1986) Stroke, 17: 472-476), and Tamura and colleagues used electrocautery to permanently occlude the MCA (Tamura, A., et al. (1981) Journal of Cerebral Blood Flow and Metabolism, 1(1): 53-60). Saleh and colleagues describe a method of permanently occluding the MCA at three locations using electrocautory (Saleh et al. (2001) Am J Physiol Regulatory Integrative Comp Physiol 281:2088-2095; Saleh et al. (2001) Am J Physiol Regulatory Integrative Comp Physiol 281:1531-1539) to study the immediate effects of permanent MCA occlusion (within 4-6 hours of occlusion). The three-point approach resulted in focal infarct areas restricted to the cerebral cortex with an associated intra-operative mortality rate of less than 1%. Chen et al. disclose a method of surgically exposing the right MCA in rats and permanently occluding it with a square knot using a suture, with or without simultaneous ligation of the right common carotid artery (CCA) or left and right CCA together (Chen, S. T. et al. (1986) Stroke, 17: 738-743). In one method, the MCA and right CCA were permanently occluded, while the left CCA was temporarily compressed by a clip. Using the latter method, mortality was lowered to 7% and infarcts were observed at a rate of 96%. However, infarct volume was quite large (100±6 mm).
While permanent occlusion of the MCA can provide a means of studying ischemic events involved in a stroke, such models do not permit examination of the effects of subsequent reperfusion.
Longa and colleagues described an animal model of ischemic stroke involving reversible occlusion of the MCA in rats (Longa, E. Z. et al. (1989). Stroke, 20: 84-91). The method involved introducing a silk suture through an incision in a terminal branch of the external carotid artery (ECA) and advancing the suture into the internal carotid artery (ICA) lumen until resistance was met, indicating that the end of the suture had passed the MCA origin and reached the proximal segment of the anterior cerebral artery (ACA), which has a smaller diameter. The incision was closed, leaving 1 cm of the suture protruding. This permitted manual withdrawal of the suture to permit reperfusion through restoration of MCA blood flow. However, due to variations in anatomy (for instance, differences in the MCA of each animal), it is difficult to place the end of the string in the same place in each animal. It is impossible to visualize and confirm the site of occlusion, the occurrence and extent of occlusion, and the extent of reperfusion. As such, the reversibly occluded animal model of Longa et al. (and other animal models based thereupon) is highly variable in terms of suture placement, occurrence of infarction, infarct size (21.9±14.5% and 25.7±13.4% for 2- and 4-hour occlusions, respectively) and neurological score of the resulting animals.
Reddy and colleagues disclosed an improvement on the method of Longa et al. (supra) which involved coating the suture with poly-L-lysine to promote adhesion of the suture end to the blood vessel endothelial lining (Reddy, M. K. and V. Labhasetwar (2009). FASEB Journal, 23: 1384-1395). However, this method suffers from many of the same disadvantages of the Longa et al. method in that the placement of the suture and extent of occlusion and reperfusion cannot be visually confirmed or assessed. The infarct volume was also relatively large (48.7±3.7 mm in control animals). Advancement of a monofilament (suture) through a vessel, as described by Longa et al. and Reddy et al. can damage the endothelium (see below).
Buchan and colleagues described a method of temporarily occluding the MCA using a microclip in male Wistar rats and spontaneously hypertensive rats (SHRs) (Buchan, A. M. et al. (1992). Stroke, 23: 273-279). In this method, the right and left common carotid arteries (CCA) were permanently or temporarily occluded through ligation, while the right MCA was surgically exposed and temporarily occluded using a microclip. Reperfusion was initiated by removing the microclip after 1 to 4 hours. This method permitted direct visual verification of MCA occlusion and reperfusion. Simultaneous occlusion of the right and left CCA and the right MCA resulted in lower variability in infarct size in SHRs. However, there was greater variability in infarct size in the healthy Wistar rats (see Buchan et al. at page 277, paragraph 1) and studies of SHR rats may not be representative. Slivka and colleagues reported permanent occlusion of the right CCA with temporary, microclip-induced occlusion of the right MCA. However, this yielded a variable infarct size in male SHRs. (Slivka, A. et al. (1995). Stroke, 26: 1061-1066; see Table 2).
Microclips are expensive and are known to damage the vascular endothelium. In some cases, adhesion of the clipped vessel occurs which results in incomplete and/or inconsistent reperfusion. Damaged endothelium (using clips or a monofilament advanced through CCA) can lead to release of endothelial factors which can increase brain damage or trauma on their own. Therefore, using such methods, it can be difficult to assess stroke damage induced by occlusion and/or reperfusion versus damage due to release of endothelial factors.
Manabe and colleagues reported a method of inducing temporary focal ischemia by temporarily occluding the right and left CCAs and the MCA (Manabe et al. (2009). Journal of Neurosurgery, electronically published Dec. 4, 2009). In this method, each CCA was exposed and a polypropylene suture was passed around a CCA and through a polyethylene tube to form a loose snare around each CCA. The MCA was exposed and temporary, three-vessel occlusion was induced by clipping the MCA with a microclip (at a point distal to the origin of the lenticulostriate arteries) and simultaneously closing the suture loops around the CCAs. The occlusion was maintained for 2 hours or 24 hours. However, the clinical relevance of the animal stroke model of Manabe et al. is questionable, as human stroke patients do not experienced occlusions of both carotids (leading to a global decrease in blood flow) in addition to occlusion of a vessel, such as the MCA.
One major hinderance of stroke research is the lack of good animal models. Questionable clinical relevance, surgery-induced damage, and/or mortality during the experimental procedure are problems in existing animal models (Gerriets T. et al. (2004). Complications and pitfalls in rat stroke models for middle cerebral artery occlusion: A comparison between the suture and the macrosphere model using magnetic resonance angiography. Stroke, 35: 2372-2377; Martin 1997). A high level of mortality during the procedure brings into question both the validity of the model and the ability of the data gleaned from the surviving animals to be representative of the group (Broderick and Hacke (2002). Treatment of acute ischemic stroke: Part 1: recanlization strategies. Circulation, 106: 1563-9). Lack of reproducibility in the size and location of the infarct produced is another problem. There remains a need for improved animal models of ischemic stroke and reperfusion injury that are more clinically relevant with highly reproducible focal infarctions, low incidence of artery damage, and low mortality rate during the experimental procedure.